Although α4-tubulin comprises only about one-fifth of the α-tubulin pool in every Drosophila egg, in the absence of α4-tubulin - in eggs of the kavar⁰/- hemizygous females - only a tassel of short microtubules forms with two barely separated daughter centrosomes. α4-tubulin is enriched in the long microtubules that embrace the nuclear envelope, and it is suggested that they push apart daughter centrosomes along the nuclear perimeter during the initial cleavage divisions. In vitro tubulin polymerization showed that α4-tubulin is required for rapid tubulin polymerization. Since tubulin polymerization is slow inside eggs of the kavar⁰/- females, only short microtubules can form within the 4 to 5 minutes allowed for the process. A tassel of short microtubules with two barely separated centrosomes forms in every egg of the Kavar^18c/+ females, in which the cytoplasm contains both wild-type and Kavar^18c-encoded α4-tubulin with an E82K amino acid substitution (E82K-α4-tubulin). E82K-α4-tubulin is incorporated into the microtubules and renders them unstable. When injected into wild-type early cleavage embryos E82K-α4-tubulin slows down the formation of long microtubules and the separation of the daughter centrosomes. Surprisingly, when injected into late cleavage embryos E82K-α4-tubulin is non-toxic. Similarly, in the neuroblasts, ectopically expressed E82K-α4-tubulin becomes incorporated into the microtubules that grow sufficiently long and function normally (Venkei, 2006).

Embryos in the animal kingdom rely almost exclusively on maternally provided molecules at the very beginning of their life. Constituents of maternal effect are synthesized by the maternal genes and are deposited into the cytoplasm of the egg cell during oogenesis for future use in embryogenesis. To identify components of the egg cytoplasm required for the commencement of embryogenesis, use was made of the 'genetic-dissection' technique, in which dominant female-sterile (Fs) mutations of Drosophila were induced and isolated that allowed the formation of normal-looking and fertilized eggs; however, embryogenesis came to an arrest shortly after fertilization. It was anticipated that, if an Fs mutation prevents the commencement of embryogenesis, the product of a wild-type (+) gene that has been identified as Fs is involved in the process and molecular analysis of its function, and would therefore shed light on the beginning of a new life (Venkei, 2006).

The Kavar^18c and the Kavar^21g Fs mutations were used to identify the αTub67C gene (full name α-Tubulin at 67C) of Drosophila (Venkei, 2005), that encodes α4-tubulin, the so-called maternal isoform of the four α-tubulin isoforms. Although eggs of the Kavar^18c/+ females appear normal and are fertilized as in wild type, embryogenesis soon comes to a standstill and a monopolar spindle appears in each of the 'die-at-start' embryos, with two nearby located centrosomes embedded in a tassel of 3-5 µm long, short and straight microtubules (Venkei, 2005). Kavar^18c has been shown to be dominant-negative (Venkei, 2005), implying that the Kavar^18c-encoded E82K-α4-tubulin molecules participate in the same process as wild-type α4-tubulin, such that E82K-α4-tubulin hinders function of wild-type α4-tubulin. It appears thus, that α4-tubulin is required for the formation of long microtubules (Venkei, 2006).

Second mutagenesis (by X-rays) of Kavar^21g led to the formation of kavar^rX21 (hereafter referred to as kavar⁰) the only known null-allele of the αTub67C gene (Venkei, 2005). A tassel of short and straight microtubules forms in every egg of the kavar⁰/- females that do not carry functional αTub67C genes (-indicates a short deficiency, which removes αTub67C and a few adjacent genes) (Venkei, 2005). The short microtubules are nucleated by two barely separated daughter centrosomes. The kavar⁰/- mutant phenotype clearly shows involvement of α4-tubulin in the formation of long microtubules and appropriate separation of the daughter centrosomes (Venkei, 2006).

In wild-type Drosophila embryo, the sperm-introduced basal body takes centrosome function and organizes the formation of long microtubules. Long microtubules compose the sperm aster and serve as route for shipment of the female pronucleus to close vicinity of the male pronucleus. They are also involved in separation of the daughter centrosomes, which migrate along the nuclear perimeter, as well as in the formation of the spindle apparatus in the early embryo. Their involvement has also been documented in the central and in the peripheral nervous systems of Drosophila embryos (Venkei, 2006).

The lack of long microtubules in eggs of the kavar⁰/- females is surprising, considering that α4-tubulin comprises only about 20% of the α-tubulin pool in the Drosophila eggs. The lack of α4-tubulin can not be substituted by another α-tubulin isoform (Matthews, 1989). Although there is plenty of α1-tubulin and α3-tubulin in every Drosophila egg - encoded by the constitutively expressed and evolutionary highly conserved αTub84B and αTub84D genes - they do not seem to support the formation of long microtubules. What makes α4-tubulin special? What is its function at the beginning of embryogenesis? Where is it localized in the early embryo? How do E84K-α4-tubulin molecules block the formation of long microtubules? This study used two mutant αTub67C alleles (Kavar^18c and kavar⁰) to understand the role of α4-tubulin in the formation and function of long microtubules. It is shown that that α4-tubulin is accumulated in the long microtubules that embrace the nuclear envelope, and it is hypothesized that the vast majority of the force that pushes apart the daughter centrosomes to opposite poles along the nuclear perimeter comes from the fast-growing interpolar microtubules in the early cleavage embryos. It appears - based on results of in vitro tubulin-polymerization assays - that α4-tubulin is required for rapid formation of long microtubules. Since the time available for tubulin polymerization is very short during the initial cleavage divisions and tubulin polymerization proceeds slowly in absence of α4-tubulin, only short microtubules form, which do not support proper centrosome separation. It is also reported that the need for α4-tubulin is limited to the initial cleavage divisions. Once the cleavage nuclei populate the egg cortex, forces other than the growing interpolar microtubules accomplish separation of the daughter centrosomes. The ectopically expressed Kavar^18c-encoded E82K-α4-tubulin molecules become incorporated into the microtubules, they do not disrupt microtubule-associated functions in imaginal disc and neuroblast cells (Venkei, 2006).

The previously described partial loss-of-function and gain-of-function mutations in the αTub67C gene showed involvement of the α4-tubulin in oocyte meiosis, cleavage mitoses, formation of the sperm aster and in the embryonic central as well as in the peripheral nervous system (Theurkauf, 1992; Matthews, 1993; Máthé, 1998; Matthies, 1999). As described in this study, complete loss-of-function of αTub67C (in eggs of the kavar⁰/- hemizygous females) leads to the formation of short and straight microtubules, suggesting that α4-tubulin is required for the formation of long microtubules. The observation is rather astonishing because α4-tubulin comprises only about 20% of the α-tubulin pool in the Drosophila eggs (Matthews, 1989) leaving plenty of α-tubulin for the formation of microtubules. What makes α4-tubulin special? α4-tubulin is a rather divergent type of the four Drosophila α-tubulins: it shares only 67% (302 of 451) amino acid identity with the evolutionary highly conserved α1-tubulin isoform (Venkei, 2006).

However, to conclude that α4-tubulin is required for the formation of long microtubules, is superficial. In vitro polymerization of tubulins revealed that, once sufficient time is available for tubulin polymerization, microtubules of the similar length will form whether α4-tubulin is present or not. It might thus be concluded that α4-tubulin is required for rapid formation of the microtubules. It appears that, although tubulin polymerization starts in eggs of the kavar⁰/- females, it goes on far too slowly and, thus, the forming microtubules can not grow sufficiently long within the time available for the process. The time allowed for tubulin polymerization is limited by the Cyclin-Cdc-controlled cyclic sets of events that start during egg activation, whether the egg is fertilized or not. The lack of formation of sufficiently long microtubules leads to arrest of the cleavage cycles and the eventual death of the embryos. Similar 'out-of-phase' and 'behind-schedule-and-lost' types of events have already been reported during Drosophila embryogenesis. For example, in embryos defective in cytoplasmic dynein-heavy-chain function the replication cycle comes to an end soon after fertilization, whereas the centrosome cycles proceed normally (Venkei, 2006).

A remarkable feature of α4-tubulin is its apparent enrichment in the so-called interpolar microtubules that embrace the nuclear envelope. The interpolar microtubules interconnect the centrosomes and were proposed to participate in separation of the daughter centrosomes (Cytrynbaum, 2003; Scholey, 2003). In fact, since diameter of the nuclei is about 10 µm up to the ninth cleavage cycle, the interpolar microtubules need to be as long as 15-16 µm, when perfect geometrical parameters during interphase is a concern. Since duration of an early cleavage cycle is about 8 minutes, of which the interphase comprises about 4 minutes, the interpolar microtubules must grow 'fast' (with a speed of about 4 µm/minute) to fulfill their functions. Along progression, beyond the ninth cleavage cycle size of the nuclei decreases to about 5 µm and, correspondingly, the interpolar microtubules need to grow to only approximately 8 µm to extend over the opposite poles. Since length of the interphase in the cleavage cycle ten is approximately 6 minutes, the slow growth rate of microtubules of 1.3 µm/minute may be adequate for the formation of sufficiently long interpolar microtubules (Venkei, 2006).